Histochemical Characterization of the Dorsal Raphe-Periaqueductal Grey Dopamine Transporter Neurons Projecting to the Extended Amygdala

The dorsal raphe (DR) nucleus contains many tyrosine hydroxylase (TH)-positive neurons which are regarded as dopa-minergic (DA) neurons. These DA neurons in the DR and periaqueductal gray (PAG) region (DA^DR-PAG neurons) are a subgroup of the A10 cluster, which is known to be heterogeneous. This DA population projects to the central nucleus of the amygdala (CeA) and the bed nucleus of the stria terminalis (BNST) and has been reported to modulate various affective behaviors. To characterize, the histochemical features of DA^DR-PAG neurons projecting to the CeA and BNST in mice, the current study combined retrograde labeling with Fluoro-Gold (FG) and histological techniques, focusing on TH, dopamine transporter (DAT), vasoactive intestinal peptide (VIP), and vesicular glutamate transporter 2 (VGlut2). To identify putative DA neurons, DAT-Cre::Ai14 mice were used. It was observed that DAT^DR-PAG neurons consisted of the following two subpopulations: TH1/VIP– and TH–/VIP1 neurons. The DAT1/TH–/VIP1 subpopulation would be non-DA noncanonical DAT neurons. Anterograde labeling of DAT^DR-PAG neurons with AAV in DAT-Cre mice revealed that the fibers exclusively innervated the lateral part of the CeA and the oval nucleus of the BNST. Retrograde labeling with FG injections into the CeA or BNST revealed that the two subpopulations similarly innervated these re-gions. Furthermore, using VGlut2-Cre::Ai14 mice, it was turned out that the TH–/VIP1 subpopulations innervating both CeA and BNST were VGlut2-positive neurons. These two subpopulations of DAT^DR-PAG neurons, TH1/VIP– and TH–/ VIP1, might differentially interfere with the extended amygdala, thereby modulating affective behaviors.